Women with non-insulin dependent diabetes mellitus, obesity and polycystic ovary syndrome experience increased pregnancy loss. All three conditions have insulin resistance in common. The mammalian blastocyst is an insulin/IGF-1 sensitive tissue. In the previous grant cycle we have shown that excess IGF-1 or insulin lead to "an insulin-resistant blastocyst." This resistance is similar to that seen in adipocytes with decreased expression of the IGF-1 receptor resulting in decreased glucose uptake via translocation of the facilitative glucose transporter, GLUT8. This decrease results in apoptosis, and miscarriage when these blastocysts are transferred back into foster mice. Our objective is to determine how an insulin-resistant state adversely affects pregnancy outcome and if insulin sensitizers like metformin have direct effects on blastocyst metabolism. We hypothesize that alterations in embryonic glucose uptake and metabolism, combined with decreased IGF-1 receptor signaling, trigger the apoptotic cascade. We also hypothesize that this insulin resistance can be partly reversed with metformin working at the level of the embryo to ameliorate the insulin signaling defect. Our rationale is that if these drugs work directly at the level of the embryo, then their use should be extended into the early pregnancy period. The following specific aims will be pursued: Specific aim 1. How does a) the decrease in insulin/IGF-1-stimulated glucose uptake or b) the decrease in IGF-1R signaling, other than that leading to glucose uptake, trigger apoptosis in the mouse blastocyst? Specific aim 2. Can both an in vitro model in trophectoderm stem cells and an in vivo model of hyperinsulinemia be used to test these hypotheses? Specific aim 3. Can direct exposure of the embryo to the insulin-sensitizing agent metformin reverse the "insulin resistance" in embryos exposed to high IGF-1. Can this also reverse the detrimental effects? At the conclusion of this proposal, we will have determined the role of IGF-1R/Akt signaling in blastocyst survival, to what extent these changes occur in vive under conditions of insulin resistance and how they are responsible for pregnancy loss. Thus, these studies are ultimately expected to have a great impact on the field of reproductive biology. [unreadable] [unreadable]